Preparation of a lipid blend and a phospholipid suspension containing the lipid blend

ABSTRACT

The present invention describes processes for the preparation of a lipid blend and a uniform filterable phospholipid suspension containing the lipid blend, such suspension being useful as an ultrasound contrast agent.

FIELD OF THE INVENTION

The present invention relates generally to processes for the preparationof a lipid blend and a uniform filterable phospholipid suspensioncontaining the lipid blend, such suspension being useful as anultrasound contrast agent.

BACKGROUND OF THE INVENTION

Manufacturing of a phospholipid contrast agent can be divided into thefollowing steps: (1) preparation of lipid blend; (2) compounding thebulk solution, which involves the hydration and dispersion of the lipidblend in an essentially aqueous medium to produce a lipid suspension;(3) filtration of the bulk solution through a sterilizing filter(s) torender the suspension free of microbial contaminants; (4) dispensing thesterile suspension into individual vials in a controlled aseptic area;(5) loading the dispensed vials into a lyophilizer chamber to replacethe vial headspace gas with perfluoropropane gas (PFP); (6) transferringthe sealed vials after gas exchange to an autoclave for terminalsterilization. There are three major obstacles in this process: (1)uniformity of the lipid blend; (2) hydration of the lipid blend; (3)uniformity and particle size of the suspension; and, (4) sterilefiltration of the suspension through a sterilizing filter(s).

Phospholipid blends are typically produced by dissolving or suspendingthe required lipids in an appropriate aqueous or non-aqueous solventsystem, and then reducing the volume either by lyophilization ordistillation. Ideally, this process produces blended solids with highcontent uniformity and purity. However, while working well on a small,laboratory scale, this simple approach is frequently problematic uponscale-up to production-size quantities. Difficulties include: (1)maintaining content uniformity during the solvent removal step (due todifferential solubilities); (2) maintaining purity (frequently a problemwhen water is used due to hydrolytic side-reactions); (3) enhancingpurity; (4) minimizing solvent volume; and (5) recovery of the finalsolids (e.g., it is not practical to scrape solids out of a largereactor).

After manufacture of a lipid blend, final compounding typically involvesintroduction of the blend into an aqueous medium. Since phospholipidsare hydrophobic and are not readily soluble in water, addingphospholipids or a lipid blend directly into an aqueous solution causesthe lipid powder to aggregate forming clumps that are very difficult todisperse. Thus, the hydration process cannot be controlled within areasonable process time. Direct hydration of phospholipids or a lipidblend in an aqueous medium produces a cloudy suspension with particlesranging from 0.6 μm to to 100 μm. Due to relatively large particle sizedistribution, the suspension cannot be filtered at ambient temperaturewhen the suspension solution temperature is below the gel-to-liquidcrystal phase transition temperatures of lipids. The lipids wouldaccumulate in the filters causing a restriction in the flow rate, and inmost cases, the filters would be completely blocked shortly after.Further reduction in the suspension particle size cannot be achievedthrough a conventional batching process, even after extended mixing(e.g., 6 hours) at elevated temperatures (e.g., 40° C. to 80° C.) with acommonly used marine propeller.

Although filtration at elevated temperatures, i.e., at above the phasetransition temperatures of lipids, is possible, a significant amount oflarger lipid particles would still be excluded when a normal filteringpressure is used. In turn, concentrations of the sterile filtrate wouldhave variable lipid content from batch to batch depending on how thelipids are initially hydrated which is in turn determined by thephysical characteristics, e.g., morphology, of the starting materials.

The process of directly hydrating the lipids or lipid blend to produce auniform suspension and filtration of the suspension through asterilization filter(s) can be difficult and costly to be scaled-up toany reasonable commercial scale, e.g., >20 L.

Thus, the presently claimed processes for manufacture of a lipid blendand the subsequent phospholipid suspension are aimed at solving theabove issues by providing a practical process that can be easily scaledand adopted to various manufacturing facilities without extensivemodification or customization of existing equipment.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide a novelprocess for preparing a lipid blend.

Another object of the present invention is to provide a novel processfor preparing a phospholipid suspension from the lipid blend.

These and other objects, which will become apparent during the followingdetailed description, have been achieved by the inventors' discoverythat dissolving a lipid blend in a suitable non-aqueous solvent prior tointroduction of an aqueous solution allows for production of aphospholipid suspension.

DETAILED DESCRIPTION OF THE INVENTION

[1] Thus, in a first embodiment, the present invention provides a novelprocess for preparing a phospholipid suspension, comprising:

(1) contacting a lipid blend with a non-aqueous solvent, whereby thelipid blend substantially dissolves in the non-aqueous solvent; and,

(2) contacting the solution from step (1) with an aqueous solution toform a lipid suspension.

[2] In a preferred embodiment, the non-aqueous solvent is selected frompropylene glycol, ethylene glycol, and polyethylene glycol 300.[3] In a more preferred embodiment, the non-aqueous solvent is propyleneglycol.[4] In another preferred embodiment, the lipid blend, comprises:

-   (a) 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine;-   (b) 1,2-dipalmitoyl-sn-glycero-3-phosphotidic, mono sodium salt;    and,-   (c) N-(methoxypolyethylene glycol 5000    carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine,    mono sodium salt.    [5] In another preferred embodiment, in step (1), the non-aqueous    solvent is heated to a temperature of about 30 to 70° C. prior to    contacting with the lipid blend.    [6] In another more preferred embodiment, the non-aqueous solvent is    heated to a temperature of about 50 to 55° C. prior to contacting    with the lipid blend.    [7] In another preferred embodiment, the ratio of lipid blend to    non-aqueous solvent is from about 5 mg of lipid blend per mL of    non-aqueous solvent to about 15 mg/mL.    [8] In another more preferred embodiment, the ratio of lipid blend    to non-aqueous solvent is about 10 mg/mL.    [9] In another preferred embodiment, in step (2), the aqueous    solution is selected from water, saline, a saline/glycerin mixture,    and a saline/glycerin/non-aqueous solvent mixture.    [10] In another more preferred embodiment, the aqueous solution is a    saline and glycerin mixture.    [11] In another more preferred embodiment, the aqueous solution is a    saline, glycerin, and propylene glycol mixture.    [12] In another more preferred embodiment, 6.8 mg/mL of sodium    chloride are present, 0.1 mL/mL of glycerin are present, 0.1 mL/mL    of propylene glycol are present, and about 0.75 to 1.0 mg/mL of the    lipid blend are present.    [13] In an even more preferred embodiment, 0.75 mg/mL of lipid blend    are present.    [14] In another more preferred embodiment, 1.0 mg/mL of lipid blend    are present.    [15] In another preferred embodiment, in step (2), the aqueous    solution is heated to a temperature of about 45 to 60° C. prior to    contacting with the solution from step (1).    [16] In another more preferred embodiment, the aqueous solution is    heated to a temperature of about 50 to 55° C. prior to contacting    with the solution from step (1).    [17] In another preferred embodiment, the process further comprises:

(3) heating the lipid suspension from step (2) to a temperature aboutequal to or above the highest gel to liquid crystalline phase transitiontemperature of the lipids present in the suspension.

[18] In another more preferred embodiment, in step (3), the lipidsuspension is heated to a temperature of at least about 67° C.[19] In another more preferred embodiment, the process furthercomprises:

(4) filtering the lipid suspension through a sterilizing filter.

[20] In another even more preferred embodiment, in step (4), thefiltration is performed using two sterilizing filter cartridges.[21] In a further preferred embodiment, in step (4), the sterilizingfilter cartridges are at a temperature of from about 70 to 80° C.[22] In another further preferred embodiment, in step (4), 0.2 μmhydrophilic filters are used.[23] In another even more preferred embodiment, the process furthercomprises:

(5) dispensing the filtered solution from step (4) into a vial.

[24] In another further preferred embodiment, the process furthercomprises:

(6) exchanging the headspace gas of the vial from step (5) with aperfluorocarbon gas.

[25] In another even further preferred embodiment, the perfluorocarbongas is perfluoropropane.[26] In another even further preferred embodiment, exchange of headspacegas is performed using a lyophilizing chamber.[27] In another even further preferred embodiment, the process furthercomprises:

(7) sterilizing the vial from step (6).

[28] In a still further preferred embodiment, in step (7), the vial issterilized at about 126-130° C. for 1 to 10 minutes.

[29] In a second embodiment, the present invention provides a novelprocess for preparing a lipid blend, comprising:

(a) contacting at least two lipids with a first non-aqueous solvent;

(b) concentrating the solution to a thick gel;

(c) contacting the thick gel with a second non-aqueous solvent; and,

(d) collecting the resulting solids.

[30] In a preferred embodiment, in step (a), the lipids are:

-   (i) 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine;-   (ii) 1,2-dipalmitoyl-sn-glycero-3-phosphotidic, mono sodium salt;    and,-   (iii) N-(methoxypolyethylene glycol 5000    carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine,    mono sodium salt.    [31] In another preferred embodiment, in step (a), the first    non-aqueous solvent is a mixture of methanol and toluene.    [32] In another preferred embodiment, in step (c), the second    non-aqueous solvent is a methyl t-butyl ether.    [33] In another preferred embodiment, in step (a), the solution is    warmed to a temperature sufficient to complete dissolution of the    lipids into the solvent.    [34] In another more preferred embodiment, in step (a), the solution    is warmed to about 25 to 75° C.    [35] In another preferred embodiment, in step (d), the solids    collected are washed with methyl t-butyl ether and dried in vacuo.    [36] In a third embodiment, the present invention provides a novel    phospholipid suspension, comprising:

(a) a lipid blend in an amount of about 0.75-1.0 mg/mL of suspension;

(b) sodium chloride in an amount of about 6.8 mg/mL of suspension;

(c) glycerin in an amount of about 0.1 mL/mL of suspension;

(d) propylene glycol in an amount of about 0.1 mL/mL of suspension; and

(e) water;

wherein the suspension is prepared by the process, comprising:

(1) contacting a lipid blend with a non-aqueous solvent, whereby thelipid blend substantially dissolves in the non-aqueous solvent;

(2) contacting the solution from step (1) with an aqueous solution toform a lipid suspension;

(3) heating the lipid suspension from step (2) to a temperature aboutequal to or above the highest gel to liquid crystalline phase transitiontemperature of the lipids present in the suspension; and,

(4) filtering the lipid suspension through a sterilizing filter.

[37] In another preferred embodiment, the lipid blend, comprises:

-   (a) 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine;-   (b) 1,2-dipalmitoyl-sn-glycero-3-phosphotidic, mono sodium salt;    and,-   (c) N-(methoxypolyethylene glycol 5000    carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine,    mono sodium salt.    [38] In another more preferred embodiment, the non-aqueous solvent    is heated to a temperature of about 50 to 55° C. prior to contacting    with the lipid blend.    [39] In another more preferred embodiment, the ratio of lipid blend    to non-aqueous solvent is about 10 mg/mL.    [40] In another more preferred embodiment, the aqueous solution is a    saline, glycerin, and propylene glycol mixture.    [41] In an ever more preferred embodiment, 0.75 mg/mL of lipid blend    are present.    [42] In another more preferred embodiment, the aqueous solution is    heated to a temperature of about 50 to 55° C. prior to contacting    with the solution from step (1).    [43] In another more preferred embodiment, in step (3), the lipid    suspension is heated to a temperature of at least about 67° C.    [44] In another further preferred embodiment, in step (4), two 0.2    μm hydrophilic filters are used.

Formulation

The present invention is contemplated to be practiced on at least amultigram scale, kilogram scale, multikilogram scale, or industrialscale. Multigram scale, as used herein, is preferably the scale whereinat least one starting material is present in 10 grams or more, morepreferably at least 50 grams or more, even more preferably at least 100grams or more. Multikilogram scale, as used herein, is intended to meanthe scale wherein more than one kilogram of at least one startingmaterial is used. Industrial scale as used herein is intended to mean ascale which is other than a laboratory scale and which is sufficient tosupply product sufficient for either clinical tests or distribution toconsumers.

Lipid blend or phospholipid blend, as used herein, is intended torepresent two or more lipids which have been blended. The lipid blend isgenerally in a powder form. Preferably, at least one of the lipids is aphospholipid. Preferably, the lipid blend contains1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC),1,2-dipalmitoyl-sn-glycero-3-phosphotidic, mono sodium salt (DPPA), andN-(methoxypolyethylene glycol 5000carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine,monosodium salt (MPEG5000-DPPE). The amount of each lipid present in theblend will depend on the desired end product. Preferred ratios of eachlipid are described in the Examples section. A wide variety of otherlipids, like those described in Unger et al, U.S. Pat. No. 5,469,854,the contents of which are hereby incorporated by reference, may be usedin the present process.

Phospholipid, as used herein, is a fatty substance containing an oily(hydrophobic) hydrocarbon chain(s) with a polar (hydrophilic) phosphorichead group. Phospholipids are amphiphilic. They spontaneously formboundaries and closed vesicles in aqueous media. Phospholipidsconstitute about 50% of the mass of animal cell plasma membrane.

Preparation of the Lipid Blend

The lipid blend may be prepared via an aqueous suspension-lyophilizationprocess or an organic solvent dissolution-precipitation process usingorganic solvents. In the aqueous suspension-lyophilization process, thedesired lipids are suspended in water at an elevated temperature andthen concentrated by lyophilization. Preferably a dissolution procedureis used.

Step (a):

The organic solvent dissolution-precipitation procedure involvescontacting the desired lipids (e.g., DPPA, DPPC, and MPEG5000 DPPE) witha first non-aqueous solvent system. This system is typically acombination of solvents, for example CHCl₃/MeOH, CH₂Cl₂/MeOH, andtoluene/MeOH. Preferably, the first non-aqueous solvent is a mixture oftoluene and methanol. It may be desirable to warm the lipid solution toa temperature sufficient to achieve complete dissolution. Such atemperature is preferably about 25 to 75° C., more preferably about 35to 65° C.

After dissolution, it may be desired to remove undissolved foreignmatter by hot-filtration or cooling to room temperature and thenfiltering. Known methods of filtration may be used (e.g., gravityfiltration, vacuum filtration, or pressure filtration).

Step (b):

The solution is then concentrated to a thick gel/semisolid.Concentration is preferably done by vacuum distillation. Other methodsof concentrating the solution, such as rotary evaporation, may also beused. The temperature of this step is preferably about 20 to 60° C.,more preferably 30 to 50° C.

Step (c):

The thick gel/semisolid is then dispersed in a second non-aqueoussolvent. The mixture is slurried, preferably near ambient temperature(e.g., 15-30° C.). Useful second non-aqueous solvents are those thatcause the lipids to precipitate from the filtered solution. The secondnon-aqueous solvent is preferably methyl t-butyl ether (MTBE). Otherethers and alcohols may be used.

Step (d):

The solids produced upon addition of the second non-aqueous solvent arethen collected. Preferably the collected solids are washed with anotherportion of the second non-aqueous solvent (e.g., MTBE). Collection maybe performed via vacuum filtration or centrifugation, preferably atambient temperature. After collection, it is preferred that the solidsare dried in vacuo at a temperature of about 20-60° C.

For the following reasons, the organic solvent dissolution-precipitationprocess is preferred over the aqueous suspension/lyophilization process:

(1) Because the lipids are quite soluble in toluene/methanol, solventvolumes are significantly reduced (relative to the aqueous procedure).

(2) Because of this increased solubility, the process temperature isalso lower relative to the aqueous procedure, thereby avoiding thehydrolytic instability of fatty acid esters.

(3) When cooled back to room temperature, the toluene/methanol solutionof lipids remains homogeneous, allowing a room temperature filtration toremove solid foreign matter.

(4) The MTBE precipitation allows quick and easy isolation of LipidBlend solids. With the aqueous process, a time-consuming lyophilizationprocess is used to isolate material.

(5) The MTBE precipitation also allows for the removal of anyMTBE-soluble impurities, which go into the filtrate waste-stream. Thispotential for impurity removal is not realized when a solution isdirectly concentrated or lyophilized to a solid.

(6) The present process affords uniform solids.

Preparation of the Lipid Suspension Step (1):

In step one, a lipid blend is contacted with a non-aqueous solvent,whereby the lipid blend substantially dissolves in the non-aqueoussolvent. Alternatively, the individual lipids may be contacted with thenon-aqueous solvent sequentially in the order: DPPC, DPPA, andMPEG5000-DPPE; DPPC, MPEG5000-DPPE, and DPPA; MPEG5000-DPPE, DPPA, andDPPC; or MPEG5000-DPPE, DPPC, and DPPA. The DPPA, being the leastsoluble and least abundant of the lipids is not added first. Adding oneof the other lipids prior to or concurrently with adding the DPPAfacilitates dissolution of the DPPA. In another alternative, theindividual lipids can be combined in their solid forms and thecombination of the solids contacted with the non-aqueous solvent.

Substantial dissolution is generally indicated when the mixture of lipidblend and non-aqueous solven becomes clear. As noted previously,phospholipids are generally not water soluble. Thus, direct introductionof a blend of phospholipid blend into an aqueous environment causes thelipid blend to aggregate forming clumps that are very difficult todisperse. The present invention overcomes this limitation by dissolvingthe lipid blend in a non-aqueous solvent prior to introduction of theaqueous solution. This allows one to evenly disperse the lipid blendinto a liquid. The liquid dispersion can then be introduced into adesired aqueous environment.

Non-aqueous is intended to mean a solvent or mixture of solvents whereinthe amount of water present is sufficiently low as to not impededissolution of the lipid blend. The amount of non-aqueous solventrequired will depend on the solubility of the lipid blend and also thefinal desired concentration of each component. As one of ordinary skillwould appreciate, the level of water present in the non-aqueous solvent,which may be tolerated will vary based on the water solubilities of theindividual lipids in the lipid blend. The more water soluble theindividual phospholipids, the more water which may be present in step(1). Preferably, propylene glycol is used as the non-aqueous solvent.However, other members of the polyol family, such as ethylene glycol,and polyethylene glycol 300 may be used.

Mechanically mixing the lipid blend and non-aqueous solvent may benecessary to achieve complete dissolution. One of ordinary skill in theart will recognize that a variety of ways of mixing are available. It ispreferred that a high shear homogenizer is used.

One of ordinary skill in the art would recognize that raising thetemperature of the solvent should aid in dissolution of the lipid blend.The temperature at which step (1) may be performed can range fromambient to the boiling point of the chosen solvent. Preferably thetemperature is from about 30 to about 70° C., more preferably about 45to about 60° C., and even more preferably about 50, 51, 52, 53, 54, or55° C. When ethylene glycol or polyethylene glycol 300 is used, it ispreferred that the temperature be from about 50 to about 60° C. and morepreferably about 55° C. Maintaining the solution at an elevatedtemperature should reduce solution viscosity and ease formulationpreparation.

A preferred procedure for dissolving the lipid blend is as follows: (a)Add propylene glycol to an appropriate weighing container. (b) Warm thepropylene glycol to about 40-80° C. in a heating bath. (c) Weigh thelipid blend into a separate container. (d) When the propylene glycol hasreached the desired temperature range, transfer the solution into thecontainer containing the lipid blend. (e) Place the container back intothe heating bath until the solution is clear. (f) Mechanically mix theLipid Blend/Propylene Glycol solution to further assure completedissolution and uniform dispersion of the lipid blend.

The ratio of lipid blend to non-aqueous solvent will, of course, belimited by the solubility of the lipid blend. This ratio will also beinfluenced by the desired amount of lipid blend in the finalformulation. Preferably, the ratio is from about 1 mg of lipid blend permL of solvent (mg/mL) to about 100 mg/mL. More preferably, the lipidblend is present in about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15mg/mL. Even more preferably, the lipid blend is present in about 10mg/mL.

Step (2):

The second step involves contacting the solution from step (1) with anaqueous solution to form a lipid suspension. The aqueous solution can bewater, saline, a saline/glycerin mixture or asaline/glycerin/non-aqueous solvent mixture. Non-aqueous solvent is asdefined previously, preferably propylene glycol. Suspension, as usedherein, is intended to indicate a dispersion in which insolubleparticles are dispersed in a liquid medium.

Once complete dissolution of the lipid blend has been achieved (step(1)), the resulting solution can then be introduced to an aqueoussolution. The aqueous solution may contain one or more componentsselected from sodium chloride, glycerin, and a non-aqueous solvent.Preferably the aqueous solution contains glycerin and sodium chloride.Preferably, a sufficient amount of propylene glycol is present in theaqueous solution, prior to addition of the solution from step 1, inorder to achieve the final desired concentration of propylene glycol.

The order of addition of desired components is not expected to seriouslyimpact the resulting lipid suspension. However, it is preferred that thelipid-blend solution is added to water, which may already contain theabove-noted additional components. Additional desired components canthen be added. It is more preferred that the lipid-blend solution isadded a solution of water and sodium chloride (i.e., saline). It isfurther preferred that the lipid-blend solution is added a solution ofwater, sodium chloride, and glycerin. It is still further preferred thatthe lipid-blend solution is added a solution of water, sodium chloride,glycerin, and propylene glycol.

It is preferred that 6.8 mg of NaCl are present per mL of formulation.Preferably, 0.1 mL of Glycerin per mL of formulation is present. A finalconcentration of 0.1 mL of Propylene Glycol per mL of formulation ispreferred. The final pH of the formulation is preferably about 5.5-7.0.The lipid blend is preferably present in an amount of 0.75-1.0 mg/mL offormulation.

The temperature of the aqueous solution can range from ambient to 70° C.Preferably, the temperature is about 45 to 60° C., with 50, 51, 52, 53,54, or 55 being even more preferred. In order to obtain completedissolution, the mixture will need to be agitated, preferably stirred.Also, the pH of the solution may need to be adjusted, depending on thedesired final formulation. Either acid (e.g., HCl) or base (e.g., NaOH)can be added to make such an adjustment.

The lipid suspension will contain liquid particles of varying sizes. Oneof the benefits of the present invention is the ability to consistentlyobtain small particles of a nearly uniform size. Thus, it is preferredthat the majority of particles obtained are less than 100 nm indiameter, more preferable less than 50 nm.

A preferred procedure for dissolving the lipid blend is as follows: (a)Add Water for Injection (WFI) into a compounding vessel. (b) Startmixing and ensure temperature is from 50-55° C. (c) Add sodium chlorideto the compounding vessel. Wait until the solid has completely dissolvedbefore proceeding to the next step. (d) Add glycerin to the compoundingvessel. Allow sufficient time for complete mixing. (e) Add the remainingPropylene Glycol that is not in the Lipid Blend/Propylene Glycolsolution. Allow time for thorough mixing. (f) Reduce mixing rate toreduce turbulence in the compounding vessel. (g) Add the LipidBlend/Propylene Glycol solution to the compounding vessel. (h) Readjustmixing to original rate. (i) Add additional WFI if necessary. (j)Continue to mix for approximately 25 minutes and assure complete mixing.(k) Verify and adjust the solution to target pH.

Step (3):

Step three involves heating the lipid suspension obtained from step (2)to a temperature about equal to or above the highest gel to liquidcrystalline phase transition temperature of the lipids present in thesolution.

One of the objects of this step is to provide a filterable suspension. Asolution/suspension is considered filterable if there is no significantreduction in flow rate within a normal process, and there is nosignificant increase in the pressure drop in the filtration system.

Experimental data indicates that the lipids in the formulation should bebeyond their gel to liquid crystalline phase transition in order tosimplify sterile filtration. When the lipids are below the phasetransition temperature, the suspension particles are rigid. However,when they are above their respective gel-liquid crystal phase transitiontemperatures, they are in a more loosely organized configuration andthus, more easily filtered.

DPPC and DPPA show phase transitions of 41° C. and 67° C. respectively.MPEG5000-DPPE is soluble in water, therefore it does not exhibit agel-liquid crystal phase transition which is characteristic of mosthydrated lipid suspensions. Because the lipids in the preferredformulation all exhibit different gel to liquid phase transitions, thehighest phase transition temperature, 67° C., is preferably used tofilter the solution. By maintaining temperature at or beyond 67° C., allthe lipids are beyond their respective phase transition, assuring theloose configuration while passing through the filters.

Heating may be achieved by jacketing the compounding vessel with a heatexchanging coil. Hot water/steam from a controlled source, e.g., a hotwater bath, or a water heater, would deliver sufficient heat to maintainthe compounding solution at a set temperature. Other heat sources knownto those of skill in the art could also be used.

Step (4):

Step four is performed by filtering the lipid suspension through asterilizing filter. The purpose behind this step being to provide asubstantially bacteria-free suspension. A filtrate is consideredsubstantially bacteria-free when the probability of the filtrate tocontain at least one colony forming microorganism is less than 10⁻⁶.

Filtration is preferably done using sterilizing filter cartridges. Also,a means of forcing the solution through the filters may be required(e.g., pumping or pressurizing). Since the solution being filtered needsto be maintained at a temperature at or above the highest gel to liquidcrystalline phase transition temperature of the lipids present in thesolution, the filtration should be performed at about this sametemperature. In order to accomplish this, the filter (e.g., sterilizingfilter cartridges) are preferably enclosed in jacketed filter housingswhich are continuously heated, e.g., by a hot water stream from atemperature controlled water bath, to ensure that the suspension isabove the lipid phase transition temperatures. The temperature of thesterilizing filter is preferably from 50 to 100° C., more preferablyfrom 60 to 90° C., and even more preferably 70, 71, 72, 73, 74, 75, 76,77, 78, 79, or 80° C.

One or more sterilizing filters may be used to filter the suspension.The required number will be based on their effectiveness at removingbacteria. It is preferred that two filters are used. The size of thefilter pores will be limited by the need to provide a bacteria-freesuspension. Preferably, 0.2 μm hydrophilic filters are used.

A bulk solution of the preferred formulation was continuously filteredthrough two 0.2 μm hydrophilic filters for up to 3 hours at a rate ofapproximately 1 liter per minute (1 L/min.), i.e., passing a total of180 liters of the suspension solution through the filters. Theexperimental results shows that there is no apparent blockage offilters. Lipid assays indicates that there is no measurable loss duringthe filtration process (due to accumulation in the filter medium).

A bulk solution of the preferred formulation was compounded at 40°C.-80° C., and the suspension was cooled to ambient temperature prior tosterile filtration. No apparent clogging of the filters were observedindicating the suspension particle size distribution is well below 0.2μm of the filter pore size. It is desirable to use heat duringfiltration in order to ensure maximum recover of the lipid blend in thesterile filtrate (i.e., to minimize potential retention of lipidparticles in the filter medium).

A preferred procedure for filtering the lipid suspension is as follows:(a) Assure all jacketed filters are at 70° C.-80° C. (b) Assure allvalves in the filtration unit are closed. (c) Connect filtration inlethose to the outlet of the compounding vessel. (d) Open valves to allowsolution to pass through the filters. (e) Flush three liters of solutionthrough the filters before collecting filtrate. (f) Continue filtrationuntil complete.

Step (5):

Dispensing the filtered solution into a vial completes step five.Preferably, this step is performed in a controlled aseptic area. One ofordinary skill in the art would recognize that the vial selected andamount of suspension delivered to the vial would depend on the end useconsidered for the lipid suspension. Dispensing can be achieved via avariety of methods, including pipette, hand-held syringe dispenser(e.g., Filamatic® syringe dispensing machine), or industrial autodispensing machine (e.g., Cozzoli or TL auto filling machine).

Step (6):

Step six is performed by exchanging the headspace gas of the vials fromstep five with a perfluorocarbon gas. A preferred method of exchange isto load the dispensed vials into a lyophilizer chamber and replace thevial headspace gas with a perfluorocarbon gas. A preferred gas isperfluoropropane (PFP). Other methods of headspace gas exchange known tothose of skill in the art may be employed.

The vials are sealed at the completion of the vial headspace gasexchange cycle. When the lyophilizer chamber pressure is brought back toatmospheric pressure by charging into the chamber with PFP. Vialstoppers are seated to seal the vials.

Step (7):

Step seven involves terminally sterilizing a vial after step six. Onemethod of terminal sterilization is through the use of an autoclave.Also, the sealed vials can be terminally sterilized in a steamsterilizer to further enhance the sterility assurance of the product.Care must be taken in the sterilization process as some degradation oflipids may be observed as a result of autoclaving. Preferably, the vialis sterilized at about 126-130° C. for 1 to 10 minutes.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

EXAMPLES

TABLE 1 Lipid Blend Target Composition Lipid Name Common Name Wt % Mole% DPPA 1,2-dipalmitoyl-sn- 6.0 10 glycero-3- phosphatidic acid,monosodium salt DPPC 1,2-dipalmitoyl-sn- 53.5 82 glycero-3-phosphatidylcholine MPEG5000 N- 40.5 8 DPPE (methoxypolyethylene glycol5000 carbamoyl)-1,2- dipalmitoyl-sn- glycero-3-phosphatidylethanolamine, monosodium salt

Lipid Blend Manufacturing Procedure

A flask is charged with toluene (3.3 L), methanol (1.2 L), DPPA (59.6g), DPPC (535 g), and MPEG5000 DPPE (405 g). After rinsing solid contactsurfaces with 0.9 L methanol, the slurry is warmed to 45-55° C. untildissolution is complete.

The solution is filtered and then concentrated in vacuo at 35-45° C. toa thick gel. Methyl t-butyl ether (MTBE, 5.4 L) is added and the mixtureis slurried at 15-30° C. White solids are collected by centrifugation orvacuum filtration, and washed with MTBE (0.9 L). The solids are thenplaced in a vacuum oven and dried to constant weight at 40-50° C. Thedried Lipid Blend is transferred to a bottle and stored at −15 to −25°C.

In another embodiment of the lipid blend manufacturing procedure of thepresent invention, the following procedure may also be used.

Alternative Lipid Blend Manufacturing Procedure

Phospholipid quantities were adjusted for purity based on a “Use As”value from the certificates of analysis. The batch size (combinedphospholipid weight) of this experiment was 2 kg.

A rotary evaporation flask is charged sequentially with toluene (3,300mL), methanol (1,200 mL), DPPA (122.9 g; corrected for “use as” purityof 97.0%), DPPC (1,098.5 g total; 500.8 g from a lot with 98.4% “use as”purity and 597.7 g from a lot with 96.7% “use as” purity), and MPEG5000DPPE (815.7 g; corrected for “use as” purity of 99.3%). After rinsingresidual solids into the flask with methanol (900 mL), the flask isplaced on a rotary evaporator (no vacuum) and the slurry is warmed tobetween 45 and 55° C. (external). After dissolution is complete, theexternal temperature is reduced to between 35 and 45° C., a vacuum isapplied, and the solution is concentrated to a white semi-solid. Theflask is removed from the evaporator and solids are broken up with aspatula. The flask is reapplied to the evaporator and concentration iscontinued. After reaching the endpoint (final vacuum pressure ² 20 mbar;white, granular, chunky solid), MTBE (5,400 mL) is added through therotary evaporator's addition tube, the vacuum is discontinued, and themixture is slurried for 15 to 45 min at 15 to 30° C. Solids are isolatedby either centrifugal or vacuum filtration, rinsed with MTBE (3,800 mL),and dried to constant weight in a vacuum oven (40 to 50° C.). Prior totransferring to polyethylene bottles with polypropylene caps, solids aredelumped through a screen (0.079 inch mesh), affording 1,966.7 g (98%)of lipid blend (SG896) as a white solid.

The preferred lipid suspension contains:

-   1,2-dipalmitoyl-sn-glycero-3-phosphotidic, mono sodium salt (DPPA);-   1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC);-   N-(methoxypolyethylene glycol 5000    carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine,    monosodium salt (MPEG5000-DPPE);-   Propylene Glycol, USP;-   Glycerin, USP;-   Sodium Chloride, USP; and,-   Water for Injection, USP.

TABLE 2 Preferred Contrast Agent Formulations Component A* B* NaCl, USP6.8 mg/mL 6.8 mg/mL Glycerin, USP 0.1 mL/mL 0.1 mL/mL Propylene Glycol,0.1 mL/mL 0.1 mL/mL USP Lipid Blend**   1 mg/mL 0.75 mg/mL  Perfluoropropane >65% >65% pH 6.0-7.0 6.0-7.0 *Formulation A has 1 mg/mLlipid blend. Formulation B has a lipid blend concentration of 0.75mg/mL. **The lipid blend is consist of 53.5 wt. % of DPPC, 6.0 wt. % ofDPPA and 40.5 wt. % of MPEG5000-DPPE.

TABLE 3 Preferred Container and Closure Component Type Vial Wheaton2802, B33BA, 2 cc, 13 mm, Type I, flint tubing vial Stopper West V504416/50, 13 mm, gray butyl lyo, siliconized stoppers Seal West 3766,white 13 mm, flip-off aluminum seals

The finished product fill volume can be from 1.0-2.0 mL/vial.

In the preparation of the preferred formulation, when the lipid blend isdirectly hydrated with the aqueous matrix solution containing water forinjection, sodium chloride, glycerin and propylene glycol, the filtrateshave less lipids as compared to the pre-filtration bulk solution. Theloss of lipids varies from 12% to 48%. These results demonstrate thatthe sterile filtration process is not effectively controlled, andtherefore, the final product lipid content is highly variable.

In contrast, using the presently described process, assay results of thelipids in show full recovery of lipids during the filtration process.Variability of assay results around the theoretical targets is withinnormal assay method variability. Particle size distribution by number,by volume and by reflective intensity of a suspension prepared by firstsolubilizing lipid blend in propylene glycol indicate that the majorityof the particles are less than 50 nm in the pre-filtered bulk solutionat 55° C. as well at 70° C. The particle distribution profile does notchange after filtration.

Utility Section

The presently claimed process is useful for preparing ultrasoundcontrast agents. Such agents should be useful for a variety of imagingapplications, including enhancing contrast in echocardiographic andradiologic ultrasound images.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise that as specifically describedherein.

1-44. (canceled)
 45. A process for preparing a lipid suspension for usewith a perfluorocarbon gas as an ultrasound contrast agent, the methodcomprising: (a) contacting phospholipids with a first non-aqueoussolvent which causes the phospholipids to dissolve and form a lipidsolution, wherein the contacting comprises the sequential addition ofindividual phospholipids to the first non-aqueous solvent wherein thephospholipids are 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine(DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid, mono sodium salt(DPPA) and N-(methoxypolyethylene glycol 5000carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine, monosodium salt (MPEG5000-DPPE), or combining said phospholipids with eachother prior to their addition to the first non-aqueous solvent; (b)contacting the non-aqueous lipid solution of (a) with a secondnon-aqueous solvent which causes the phospholipids to precipitate out asa solid lipid blend; (c) collecting the solid lipid blend; (d)contacting the solid lipid blend with a third non-aqueous solvent whichcauses the lipid blend to dissolve to form a lipid blend solution; (e)contacting the lipid blend solution with an aqueous solution to yieldthe lipid suspension; and (f) filtering the lipid suspension through oneor two sterilizing filters to form a filtered lipid suspension.
 46. Theprocess of claim 45, wherein the one or two sterilizing filters are 0.2micron filters.
 47. The process of claim 45, further comprisingdispensing the filtered lipid suspension into a vial.
 48. The process ofclaim 47, further comprising exchanging the headspace gas of the vialwith the perfluorocarbon gas.
 49. The process of claim 48, wherein theperfluorocarbon gas is perfluoropropane.
 50. The process of claim 48,further comprising sterilizing the vial.
 51. The process of claim 50,wherein the vial is sterilized at about 126-130° C. for 1 to 10 minutes.52. The process of claim 45, wherein (i) the first non-aqueous solventis a mixture of methanol and toluene; (ii) the second non-aqueoussolvent is methyl t-butyl ether; (iii) the third non-aqueous solvent ispropylene glycol; (iv) the aqueous solution comprises sodium chloride,glycerin, and propylene glycol.
 53. The process of claim 52, wherein (i)the third non-aqueous solvent is heated to a temperature of about 50-60°C. prior to contacting with the solid lipid blend; (ii) the aqueoussolution is heated to a temperature of about 45-60° C. prior tocontacting with the lipid blend solution.
 54. The process of claim 47,wherein (i) the first non-aqueous solvent is a mixture of methanol andtoluene; (ii) the second non-aqueous solvent is methyl t-butyl ether;(iii) the third non-aqueous solvent is propylene glycol; (iv) theaqueous solution comprises sodium chloride, glycerin, and propyleneglycol.
 55. The process of claim 54, wherein (i) the third non-aqueoussolvent is heated to a temperature of about 50-60° C. prior tocontacting with the solid lipid blend; (ii) the aqueous solution isheated to a temperature of about 45-60° C. prior to contacting with thelipid blend solution.
 56. The process of claim 48, wherein (i) the firstnon-aqueous solvent is a mixture of methanol and toluene; (ii) thesecond non-aqueous solvent is methyl t-butyl ether; (iii) the thirdnon-aqueous solvent is propylene glycol; (iv) the aqueous solutioncomprises sodium chloride, glycerin, and propylene glycol.
 57. Theprocess of claim 56, wherein (i) the third non-aqueous solvent is heatedto a temperature of about 50-60° C. prior to contacting with the solidlipid blend; (ii) the aqueous solution is heated to a temperature ofabout 45-60° C. prior to contacting with the lipid blend solution. 58.The process of claim 49, wherein (i) the first non-aqueous solvent is amixture of methanol and toluene; (ii) the second non-aqueous solvent ismethyl t-butyl ether; (iii) the third non-aqueous solvent is propyleneglycol; (iv) the aqueous solution comprises sodium chloride, glycerin,and propylene glycol.
 59. The process of claim 58, wherein (i) the thirdnon-aqueous solvent is heated to a temperature of about 50-60° C. priorto contacting with the solid lipid blend; (ii) the aqueous solution isheated to a temperature of about 45-60° C. prior to contacting with thelipid blend solution.
 60. The process of claim 50, wherein (i) the firstnon-aqueous solvent is a mixture of methanol and toluene; (ii) thesecond non-aqueous solvent is methyl t-butyl ether; (iii) the thirdnon-aqueous solvent is propylene glycol; (iv) the aqueous solutioncomprises sodium chloride, glycerin, and propylene glycol.
 61. Theprocess of claim 60, wherein (i) the third non-aqueous solvent is heatedto a temperature of about 50-60° C. prior to contacting with the solidlipid blend; (ii) the aqueous solution is heated to a temperature ofabout 45-60° C. prior to contacting with the lipid blend solution.